Fibronectin is a ubiquitous extracellular glycoprotein containing around 5% carbohydrate. It exists in a soluble form in body fluids and in an insoluble form in the extracellular matrix. Fibronectin plays a major role in many important physiological processes, such as embryogenesis, hemostasis, thrombosis and wound healing (Potts, J. R. and Campbell, I. D. Current Opinion in Cell Biology 6:648–55, 1994). The characteristic form of plasma fibronectin is a disulfide-bonded dimer of 440,000 daltons, each subunit having a molecular weight of about 220,000 daltons. Plasma fibronectin is also known by various other names, including cold-insoluble globulin, antigelatin factor, cell attachment protein, cell spreading factor, and opsonic alpha 2-surface binding glycoprotein. These names reflect biological activities of fibronectin such as cell recruitment, opsonization of particulate debris, and promotion of wound contraction. Reviews on structure and activities of fibronectin have been published elsewhere (Hynes, R. O. Fibronectins, Rich, A., ed. New York, Springer-Verlag 1990).
Wound healing is usually divided into three phases: the inflammatory phase, the proliferative phase, and the remodeling phase. Fibronectin has been reported to be involved in each stage of the wound healing process, particularly by creating a scaffold to which the invading cells can adhere. Initially, there is a release of many mediators to the wound site such as fibronectin and fibrinogen. Fibronectin promotes inflammatory cell migration into the wound and debris phagocytosis by monocytes. Thereafter, angiogenesis and reepithelialization take place. At this stage, fibronectin exerts chemotactic activity on endothelial cells, and promotes epithelial cell and fibroblast migration onto the basal membrane. Fibronectin also appears to be an essential component of the remodeling phase where it plays a major role in the organization of collagen fibrils. The fibrillar collagen ultimately forms fibrous bundles that greatly enhance the tissue tensile strength, leading to wound closure. Normally found in plasma at a concentration of about 300 μg/mL, fibronectin is extracted and purified using a method developed by Horowitz and Chang (Horowitz, B. and Chang, M. D. Y. “Preparation of fibronectin for therapeutic administration in Fibronectin, D. F. Mosher ed., Academic Press, San Diego 441–455 (1989)).
Topically applied plasma fibronectin has been reported as being useful for increasing the rate of wound healing such as in corneal wounds (Nishida, T. et al., Japan Journal of Ophthalmology 26: 416–24, 1982; Phan, T. M. et al., American Journal of Ophthalmology 104:494–501, 1987) and leg ulcers (Wysocki, A. et al., Arch. Dermatology 124: 175–177, 1988). However, there is no suitable topical carrier for use in treating wounds that can ensure delivery of an effective amount of fibronectin in a pharmaceutically acceptable formulation. A major limiting factor in developing an effective topical dosage form of a drug is not only having an active drug, but also having a formulation that allows the passage of the active drug from the carrier into a site of delivery.
A topical formulation, which maximizes the contact time of fibronectin to the wound and controls the release of fibronectin into the wound, is a hydrogel formulation. In drug delivery, the term hydrogel is typically reserved for polymeric materials that can absorb a significant amount of water (>20% of its dry weight) while maintaining a distinct three-dimensional structure (Gehrke, S. H. and Lee, P. I., “Hydrogels for drug delivery systems,” In Specialized Drug Delivery Systems Manufacturing and Production Technology, Chapter 8, Vol. 8, PP 333–392, Marcel Dekker, New York 1990). The most important characteristic of a hydrogel is its degree of swelling in water. Hydrogels mimic living tissue more closely than any other non-natural material. Their immediate resemblance to tissue is in their soft, flexible nature and high water content. This helps minimize mechanical irritation and damage to body tissues. Other advantages of hydrogel formulations include: ability to keep the wound moist which results from their high water content, ability to absorb excess water (exudate) in the wound, ease of application to and removal (by washing) from the wound. They also provide a cool feeling when topically applied, a property that can increase patient comfort.
Hydrogels have four major properties: swelling degree, biocompatibility, permeability and swelling kinetics. Example of such compounds include vinyl polymers (e.g. polyacrylic acid), cellulose and cellulose derivatives. Polyacrylic acid polymer, also referred to as carbomer, e.g. Carbopol® carbomer (BF Goodrich), was chosen over other polymers (e.g. cellulose and cellulose derivatives), because it was shown to be superior to other pharmaceutically acceptable formulations in the delivery of fibronectin to skin wounds.
Hydrogel formulations comprising a water soluble, pharmaceutically acceptable polymer which can include increasing concentrations of fibronectin are described in U.S. Pat. No. 5,641,483, entitled “Wound Healing Formulations containing Human Plasma Fibronectin”, which is incorporated by reference herein in its entirety. Methods for preparing non-buffered aqueous concentrated solutions of fibronectin and hydrogels containing up to 1% of fibronectin are described in U.S. Pat. No. 5,821,220, entitled “Method of Producing Concentrated Non-Buffered Solutions of Fibronectin” and International Application No. PCT/CA97/00966, International Publication No. WO 98/26797, entitled “Wound Healing Formulations Containing Human Plasma Fibronectin”, both of which are incorporated herein by reference in their entirety.
Alginates are naturally occurring substances extracted from marine brown algae and used in the pharmaceutical, cosmetic, textile and food industry. Alginates are polyanionic polysaccharides composed of linear binary copolymers of D-mannuronic acid and L-guluronic acid. The most common uses are based on the polyelectrolytic nature of the alginates, which provides the basis of their gelling properties and their ability to swell. The commercially available sodium alginates are water soluble. When such alginates are added to a solution containing polyvalent ions, for example bivalent alkaline earth metal ions such as Ca++, alginate gels having a semi-solid form are produced. This is a result of a ionic crosslinking of several alginate chains.
Calcium alginates have long been known for their ability to form fibres or nonwoven materials. These have been used primarily as swabs or dressings for medical, surgical or other purposes, such as described in European Patent Specification, EP 0721355 B1, entitled “Alginate Wound Dressings, which is incorporated herein by reference in its entirety. Supplied in the form of nonwoven wound dressings for the treatment of exudating wounds, the calcium alginate dressing is said to encourage the formation of controlled ion-active gel over the wound site which reacts with the sodium ions in the exudate. Examples of exudative wounds include pressure ulcers, venous stasis ulcers, diabetic ulcers, arterial ulcers, second degree burns and skin graft donor sites.